Development of Spectrophotometric Method to Assay Sulfadiazine in Pure and in Pharmaceutical Dosage form through Diazotization and Coupling Reaction

This study involves the development of a sensitive accurate and low-cost spectrophotometric procedure to analyze sulfadiazine as pure and in its dosage forms. In this method sulfadiazine was diazotized with an equimolar solution of sodium nitrite in the presence of hydrochloric acid to form corresponding diazonium salt followed by coupling with phloroglucinol reagent in basic solution of NaOH to produce a yellow water-soluble azo dye that has maximum absorption at 417 nm versus reagent blank. Under optimum conditions. The linearity of the method obeyed Beer’s law in the concentration range 0.25-15 μg/ml with an excellent determination coefficient (R 2 = 0.9992) and molar absorptivity 3.81×10 4 l/mol.cm. The detection limit (LOD) and quantification limit (LOQ) have also been estimated and their values were found to be 0.192 and 0.563 μg/ml, respectively. A relative error% was calculated and found in the range -2.75% to 4.02%, while the precision (RSD%) was estimated as ≤ 1.36%. The stoichiometry of the resulting azo dye was found to be 1:1 sulfadiazine: with stability constant 0.37×10 7 l/mol. The suggested procedure was applied to the analysis of sulfadiazine in cream and veterinary drugs (powder and injection).


INTRODUCTION
Sulfonamides are a class of drugs that have been commonly used as antibacterial agents in veterinary practice and in medicine especially in the treatment of eye infections and urinary tract infections as a prophylaxis of rheumatic fever (Petri, 2001). Sulfadiazine (SDZ), is one of the common sulfonamide's groups in clinical practice, chemically it is [4-amino-N -pyrimidin-2-ylbenzenesulfonamide], (John and John, 2004), whereas its chemical structure is: C 10 H 10 N 4 O 2 S M.Wt. =250.3g/mol SDZ is a synthetic agent that has been used in human and veterinary therapy about 60 years (Pecorelli et al., 2004) . It eliminates bacteria that cause infections by inhibiting the formation of folic acid inside the cell of bacteria (Vandana et al., 2011). SDZ has been employed to those prophylaxis furthermore in the medication of contamination for parasitic protozoa called toxoplasmosis gondii and also in the medicine for contamination brought about because of pneumocystis carinii, an extracellular lung pathogen, which makes pneumonia for immunecompromised hosts. (Vree et al., 1995). The literature survey revealed that several methods have been reported for direct or indirect estimation of SDZ, they include: potentiometry (Ayman et al., 2009), Nanozyme-labeled biomimetic immunoassay (He et al., 2020), capillary zone electrophoresis (Berzas et al., 2001), flow injection-flame atomic absorption (FI-FAAS) device (Haji Shabani et al., 2011), tandem mass spectrometric detection, (Forti and Scortichini, 2009), ICPatomic emission spectroscopy (Xiao-Ling et al., 2010) and immune-chromatographic assay (Wang et al., 2007). However, most of these methods either require several manipulation steps, sophisticated, cloud point extraction and expensive instruments which may not be available in every laboratory or they had high detection limit.
Several important spectrophotometric methods have also been employed to assay of SDZ in bulk and in its pharmaceutical preparations. Some of these methods based on the diazotization coupling reaction by using various organic coupling reagents such as, histidine (Othman and Kadder, 2006), oxine (Naik et al., 2007), pyrogallol (Naser et al., 2018), 2,6dihydroxybenzoic acid (Mohammed and Zebary, 2013) and 4-amino-2-hydroxy acetophenone (Al-Rufaie, 2016). Other methods are either based on the oxidative coupling reactions of SDZ with p-N,N-diethylphenylenediamine sulphate using KIO 4 as oxidizing agent (Nagaraja et al. , 2010) . In addition, determination of SDZ via charge transfer complex formation with phenosaphranine (Al-Attas, 2003) have been also reported. SDZ was also estimated through the oxidation of SDZ with permanganate solution in alkaline medium producing bluish-green colour of manganate MnO 4 2appeared absorption peak at 610 nm (Siddiqui et al., 2013). In the present investigation, a phloroglucinol (Phl) reagent has been employed to develop a simple , fast and inexpensive visible spectrophotometric method for the assay of SDZ in pure form and in its pharmaceutical drugs. This method was based on the reaction of Phl reagent with diazotized SDZ to yield a water-soluble yellow dye in a basic solution that has been employed to assay of SDZ in burn cream, powder and veterinary injectable solution.

EXPERIMENTAL Apparatus
A double beam UV-visible spectrophotometer (JASCOV-630) with 1.0-cm quartz cells was used for all absorbance measurements and absorption spectra. While a professional Benchtop pH meter BP3001 was used for the pH measurements.

Chemical Reagents
All chemical substances used were of the highest purity available and were obtained from Fluka, BDH and Merk companies. The standard material of SDZ was provided from the state company for drug industries and medical appliances (SDI), Samarra-Iraq.
-SDZ Stock Standard Solution 500 μg/ ml: A 0.0500 g of pure SDZ was dissolved in about 5 ml of ethanol and the volume was then made up to 100 ml with distilled water in a 100 ml calibrated flask. This solution was kept in a dark bottle. Similarly, a working standard solution (50 μg /ml = 1.9976 × 10 -4 M) of SDZ was freshly prepared by diluting 10 ml of the stock solution with distilled water in a 100 ml calibrated flask (Mohammed and Zebary, 2013). Phloroglucinol (Phl) Solution 0.1% w/v: A 0.100 g of Phl reagent was dissolved in small portions of distilled water and the volume was made to 100 ml in a calibrated flask with the same solvent. The solution was then transferred to a dark bottle. Sodium nitrite stock solution 1.9565 × 10 -3 M: This solution was prepared by dissolving 0.0135 g of NaNO 2 in 100 ml distilled water using a volumetric flask. Similarly, a working solution 1.9976 ×10 -4 M of NaNO 2 was then prepared by diluting 10.2 ml of the stock solution with distilled water in a 100 ml calibrated flask.

Recommended Procedure and Calibration Curve
Into a series of 20 ml calibrated flasks, 0.1 to 7 ml of 50 μg/ml pure SDZ solution, an equimolar of 1.9976×10 -4 M NaNO 2 and 1ml of 1M hydrochloric acid solutions were added. The contents were mixed thoroughly and kept constant at room temperature for 5 min then followed by adding 1.5 ml of 0.1% Phl reagent and 2 ml of 1M sodium hydroxide solutions. The contents were Shaked well and made up to the mark with distilled water. The absorbance of the azo dye formed was recorded at 417 nm against blank solution that was prepared similarly but without drug. A linear relationship between absorption and SDZ concentration in the range 5 300 μg of SDZ/20 ml was obtained Fig. (1). The apparent molar absorptivity and Sandell's sensitivity were found to be 3.81 ×10 4 l/mol.cm. and 0.00657 μg/cm 2 , respectively.

Recommended Procedure to Assay SDZ in Drug Injection solution:
The injection container (each ml of injection contains 200 mg SDZ and 40 mg trimethoprim, TMP) was mixed well to get a homogeneous solution. A 1ml of the injection solution was diluted with distilled water in 100 ml calibrated flask to prepare 2000 μg/ml SDZ solution and similarly a 10 ml of it was pipetted into a 100 ml calibrated flask and with distilled water made up to the mark (this is stock solution). A working solution of injection containing 50 μg/ml of SDZ was prepared by diluting 25 ml of the stock solution with distilled water using 100 ml calibrated flask. An aliquot of the diluted drug solution was then treated as done in recommended procedure. Flamalife Cream (each 100 g cream contains 1g silver SDZ): A 1.0 g of flamalife cream was dissolved in 5 ml of ethanol and transferred to 50 ml ether, shake well and moved to 50-ml isolating channel. SDZ was then removed multiple times with 25 ml refined water. The watery layer was gathered, separated and finished to 100 ml with refined water in utilizing volumetric carafe (United States Pharmacopeia, 1995). Each mL of this solution containing 100 μg SDZ. An aliquot of diluted solution of the drug was then analyzed using the recommended procedures.

Powder:
An accurately weighed 0.025g of the drug powder (each 1g powder contains 400 mg SDZ and 80 mg TMP) was dissolved in about 10 ml of distilled water. The resulting solution was then mixed well, filtered and diluted to 100 ml with distilled water in a calibrated flask. Each ml of this solution contains 100 μg of SDZ. The recommended procedure was then followed for the determination of SDZ by taking an aliquot of diluted solution of the drug.

RESULTS AND DISCUSSION Preliminary conditions
Under the response condition, SDZ was reacted with an equimolar of NaNO 2 in acidic solution to give the relating diazonium salt, which goes through a diazotization procedure with Phl as a coupling agent to produce the azo dye (Scheme 1): sulphadiazine diazotized sulfadiazine

Scheme 1
The azo dye gave maximum absorption at 417 nm against blank solution. The dye intensity has been found to be proportional to the SDZ amount that originally present in the solution.

Optimum Conditions for the Reaction Effect of Different Amounts of Various Acids on the Absorbance
The reaction of diazotisation requires acid solution. Therefore, the effect of different amounts of various acidic solutions (1M) such as, HCl, H 2 SO 4 , HCOOH, HNO 3 and CH 3 COOH has been examined on the dye colored. The experimental results are shown in Fig. (2). The data in Fig. (2) illustrate that HCl was convenient as acidic medium for a maximum stability and sensitivity and 1ml of 1M HCl with occasionally shaking for 5 and 6 min were found to be suitable, therefore 5 min was selected for the subsequent investigation. The results are explained in Fig. (3).

Effect of Phl Amount
The influence of various amounts (0.5-2.0) ml of Phl reagent (0.1%) on the azo dye absorbance has been investigated. The results in Fig. (4) indicated that 1.5 ml of Phl (0.1%) are the most suitable volume to give high absorbance and good determination coefficient value (R 2 =0.9995) therefore, it was selected for coupling reaction. Absorbance µg of SDZ /20 ml

Fig. 4: Effect of reagent amounts on absorbance
The effect of time on the completion of the coupling reaction of diazotized SDZ with Phl reagent has been studied by allowing the solution of the reactants after the addition of Phl solution to stand at room temperature for different times Fig. (5) then the other reagents were added and the absorbance was recorded at 417 nm against the reagent blank.  5) reveals that the coupling reaction was complete immediately after mixing the diazotized SDZ with Phl reagent and the absorbance remained maximum and stable for at least 7 min. at room temperature.

Effect of alkaline solution
The effect of different amounts 0.5-3 ml of various alkaline solutions (1M) such as, sodium hydroxide, sodium carbonate, sodium bicarbonate and potassium hydroxide has been examined for the purpose of producing azo dye with intense colour and lower blank value. The results in Fig. (6) revealed that maximum sensitivity and stability were obtained only when the reaction was carried out in the presence of sodium hydroxide solution. While, sodium carbonate and sodium bicarbonate exhibited low sensitivity which may be due to pH variation. Therefore, 2.0 ml of 1M sodium hydroxide at (pH=12.1) was found to be optimum.

Development Time and Stability Period
Under the optimal experimental conditions, the influence of time on the stability of the colored azo dye at 417 nm has been carried out by preparing two different amounts 50 and 150 μg of SDZ. The absorbance was measured at dissimilar pauses of time up to 60 minutes. The results in Fig.(7) illustrate that the absorbance of azo dye reached maximum value after the reaction mixture solution was mixed and the absorbance remained stable for at least 60 minutes at room temperature.

Absorption Spectrum
Under the optimum reaction conditions, SDZ was reacted with equimolar of NaNO 2 solution in the presence of HCl solution to give the corresponding diazonium salt. The diazonium salt was then coupled with Phl reagent in a basic solution of NaOH. A yellow water-soluble azo dye was obtained which showed maximum absorption at 417 nm versus the colorless reagent blank Fig. (8). The intensity of the azo dye formed was found to be proportional to the amount of SDZ originally present in the solution.

Fig. 8: Absorption spectra of 50 ppm SDZ treated according to the recommended procedure versus (A) reagent blank , (B) distilled water and (C) blank solution measured versus distilled water Quantification
The limits of Beer′s law, molar absorptivity (ε max ), accuracy (recovery%), precision (RSD%), conditional stability constant (Hargis, 1988), LOD and LOQ values were calculated and the data are summarized in (Table 1), which indicates that the proposed method is sensitive, precise and accurate. Linearity was represented by the regression equation and the corresponding determination coefficient (R 2 ) for SDZ determined by the proposed method represents the excellent linearity.

Composition of the Azo Dye
The stoichiometry of the product was investigated under the established conditions by applying the continuous variation (Job's) and the mole ratio methods (Delevie, 1997). In continuous variation method, volumes 0.5-4.5ml of 1.9976×10 -4 M portions of SDZ (VS) were diazotized using equimolar of 1.9976×10 -4 M sodium nitrite and 1 ml of 1M HCl and coupled according to the recommended procedure with the corresponding complementary volume of 1.9976×10 -4 M Phl solution (VR) to give a total volume of 5 ml for (VS+VR) in 2 ml of 1M NaOH and diluted to 20 ml with distilled water. In mole ratio method, increased volumes 1-4 ml of 1.9976×10 -4 M Phl solution (VR) were added to a 1 ml of 1.9976×10 -4 M of SDZ (VS) which was diazotized by using 1 ml of 1.9976×10 -4 M sodium nitrite in presence of 1ml of 0.8 M HCl, 2 ml of 1M NaOH were added and the absorbance was recorded at 417 nm after dilution to the mark with distilled water. The results obtained in Fig. (9) are revealed that the azo dye was formed by a 1:1 combining ratio of diazotized SDZ to Phl. The structure of the azo dye according to the results obtained in Fig. (9), can be represented as follows Fig. (10):

Application of the Method
The present method was applied successfully to the analysis of SDZ in various pharmaceutical preparations containing SDZ (cream, veterinary powder and injection). The results are summarized in (Table 2) reveal that the proposed procedure is in good agreement and with the declared content.

Evaluation of the suggested procedure
Standard additions method was followed to check the validity of the suggested procedure which proves that the recommended method can be successfully applied for determining SDZ without interferences. The data are shown and listed in Fig. (11) and (Table 3).  Table 3: The results of standard addition methods for analysis of SDZ CONCLUSION The proposed method for the determination of sulfadiazine in pharmaceutical samples have the advantage to be sensitive, inexpensive and simple since it does not need neither temperature control nor solvent extraction step. The method is also accurate and precise enough to be successfully adopted as an alternative to the existing spectrophotometric method and evaluation of drugs in pharmaceutical preparations.